The invention relates generally to the fields of Raman spectroscopy and microscopic imaging of biological cells. The invention also relates to dynamic chemical imaging in general.
Understanding what is occurring, particularly on a molecular scale, in and among biological cells permits one to understand the behavior of the cells and influence their behavior in desired ways. For instance, most drugs exert their pharmacological effects based on their effects on cells. However, it has been difficult to understand the effects of a drug on an individual cell or small group of cells on a biochemically-relevant scale. Instead, macroscopic effects of a drug on a tissue or an entire organism have been observed and extensive experimentation and educated guessing has been required to understand the biochemical basis of a drug's effect. A significant need exists for methods of better observing the interactions of cells with each other and with the chemicals and phenomena (e.g., temperature or fluid shear stress) that occur in their environment. The present invention satisfies this need by providing a method of dynamically imaging chemical and biological systems on a scale relevant to understanding behavior and characteristics of individual cells and their subcellular components.
Jian Ling and colleagues have described a microscopic system for obtaining limited Raman spectral information from cells. See, for example Ling et al., 2002, Appl. Optics 41(28):6006-6017; U.S. Provisional Application 60/189,123, filed 14 Mar. 2000; U.S. Non-provisional application Ser. No. 09/804,774; and U.S. Non-provisional application Ser. No. 10/750,603. The Applied Optics manuscript describes a univariate method purportedly used to prepare images showing distribution of paclitaxel in breast cancer cells. The methods involve combining an optical image of a cell with a image of the cell prepared by subtracting combined Raman scattered and fluorescently-emitted radiation at 1080 cm−1 (a Raman shift (RS) value at which scattering attributable to paclitaxel is purported to be insignificant) from combined Raman scattered and fluorescently-emitted radiation at 1000 cm−1 (an RS value at which paclitaxel is purported to exhibit a peak).
It is unclear whether the data of Ling et al. support their contention that they are observing paclitaxel in breast cancer cells. For example, they obtain a Raman spectrum of paclitaxel in powdered form (FIG. 3 in the paper, FIG. 1 in the patent applications) and demonstrate that scattering at 1002 cm−1 is much greater than scattering at 1080 cm−1. A Raman spectrum of paclitaxel+ethanol+CREMAPHOR (RTM)+PBS (FIG. 4 in the paper) purportedly shows that the paclitaxel Raman peak at 1002 cm−1 is preserved (but shifted to 1000 cm−1) in this mixture and that there is little or no RS for paclitaxel at 1080 cm−1. The figure shows a small peak at 1000 cm−1 and a possibly broad peak or shoulder at 1080 cm−1, but there is no data from which to determine what, if any, contribution paclitaxel makes to the 1080 cm−1 characteristic. The figure is also not corrected for fluorescent emission.
Even assuming that Ling et al. were able to identify paclitaxel in solution in FIG. 4 of the paper, it is not clear what the Raman spectrum of paclitaxel would look like when it is associated with a cell or a component of a cell, such as a microtubule. In short, it is not clear that the Raman signal that Ling et al. observe in paclitaxel-treated cells can, in fact, be correlated with the presence of paclitaxel.
Comparing the Raman images (center column in FIG. 8 of the Ling paper), it remains unclear what is being imaged, although at least some of whatever is being imaged is clearly shown (first row) to be present prior to treatment of the cells with paclitaxel. It is uncertain whether paclitaxel binding with microtubules would be expected to be as clumped as the Raman-active entities in the pictures in FIG. 8 appear to be. Microtubules are often substantially broadly spread throughout the cell, except during mitosis.
The univariate method of analyzing Raman scattering purportedly caused by paclitaxel may, even if it is valid under the circumstances purportedly present in the mixture studied by Ling et al. (i.e., a single strong Raman peak purportedly corresponding to paclitaxel and the particular deconvolution scheme), be limited in its utility to the particular conditions present in their experimental system. Such univariate analysis is unlikely to be broadly applicable. For instance, it is an inappropriate methodology to use for analysis of systems that exhibit more complex Raman spectra than that purportedly exhibited by the system described by Ling et al.
Another shortcoming of the optical systems used by Ling et al. is that it employs a rotating dielectric bandpass filter system to select Raman shift values for analysis. This system requires physical rotation to analyze different Raman shift values, preventing rapid analysis of multiple Raman shift values. Furthermore, rotation of the system displaces the image, requiring realignment of the Raman and optical images.
Sharonov et al. (1994, Analytica Chimica Acta 290:40-47) describe a cellular imaging system that relies on assessment of fluorescent emissions from cells and/or compounds in the cellular milieu. A significant drawback of imaging systems that rely on detection of fluorescence is that such systems exhibit relatively low spectral resolution, owing to the broad spectral width of most, if not all, fluorescent emissions. Another drawback is that many molecules and cellular components of interest do not fluoresce. Such molecules and components cannot be fluorescently imaged unless labeled—a process which can alter the behavior and characteristics of the molecule or component. Furthermore, live and dead cells often exhibit intense fluorescence background emissions that can interfere with fluorescent observation of a target of interest. Because most fluorescent emissions are not specific for the molecular species emitting the radiation, such background can complicate or prevent efforts to meaningfully interpret fluorescence.
U.S. Pat. No. 5,784,162 discloses methods of quantitatively detecting components in three dimensions in an environment containing cells or tissues. The patent discloses spectral imaging methods. The methods involve combining spectroscopic and imaging data. Spectral unmixing techniques are described in the context of fluorescence microscopy. The systems involve use of an optical path having a physically rotatable element for modulating optical interference. The system uses a stationary detector that is asserted to exhibit one-to-one correspondence with areas of the sample being imaged. This patent describe spectral imaging, rather than chemical imaging. Spectral imaging is low resolution and cannot spectrally resolve individual molecular species. The technology described in this patent is appropriate for analysis of large signals, such as fluorescent emissions, but is inappropriate for analysis of weaker signals, such as Raman scattered radiation.
U.S. Pat. No. 6,070,583 discloses methods of fluorescence and Raman imaging in two and three dimensions. The methods rely on temporally resolving inelastically scattered radiation assessed at multiple detection points surrounding an illuminated point to determine the distance of the scattering body from each of the detection points. Two- and three-dimensional representations of the illuminated system can be constructed from these data. The patent discloses that Raman spectral characteristics of scattered light can be used to characterize the chemical identity of the scattering body. The patent does not disclose whether the methods can be used to detect scattering bodies smaller than tissue lesions. Furthermore, the patent does not disclose combining spatial resolution data with optical imaging data, since the patent is directed to planar tomographic analysis of macroscopic tissues.
Chemical imaging is known in the art. One example of an apparatus used for chemical imaging is taught in U.S. Pat. No. 6,002,476, entitled “Chemical Imaging System,” to Treado et al. Among other things, U.S. Pat. No. 6,002,476 teaches the use of Raman chemical imaging for analysis of a static sample, e.g., for assessing whether a particular tissue sample corresponds to normal tissue or breast cancer tissue. Other chemical imaging systems for assessment of static samples exist in the art.
In contrast to the prior art, the present invention uses chemical imaging to assess and observe non-static samples (i.e., samples that vary over time). Among other things, the present invention may be used to detect dynamic changes that occur in the sample over an observation period.